Transcriptional regulation of Ard1b, a testis-predominant isoform of the catalytic subunit of mouse N-alpha terminal acetyltransferase, and the role of protein N-alpha acetylation Investigators: Pang, Fang, Clark, Rennert;in collaboration with Chan, Taft We have cloned a novel Ard1a (Arrest defective 1) gene homolog, known as Ard1b, that demonstrated testis specificity and was highly expressed during male meiosis in the mouse. We have shown Ard1b is functionally equivalent to Ard1a in the reconstitution of N-alpha-acetyltransferase activity in vitro. The testis-specific expression of Ard1b indicates its transcription is suppressed in somatic tissues. We subsequently identified Ard1b transcription is regulated epigenetically by DNA methylation: reactivation of Ard1b transcription occurs after treatment with 5-aza-deoxycytidine in mouse cells that do not express the gene. The two CpG islands located at the 5 end of Ard1b gene are hypermethylated in mouse somatic tissues, but hypomethylated in mouse testicular germ cells. We characterized the promoter region of the Ard1b gene. Reporter assays on different regions upstream of the gene led to identification of two upstream genomic regions that may contain inhibitory and enhancer element(s) for Ard1b transcription. We found the core promoter sequence for Ard1b is localized to a region spanning from -148 to at least +150 base-pairs with respect to the transcriptional start site of the gene, a region which is hypermethylated in cells that do not show Ard1b expression. Within this region, we predicted the presence of binding sequences for several transcription factors. By gene over-expression and knockdown experiments, we confirmed the role of Specificity protein 1 (Sp1) in the activation of Ard1b transcription. Similar experiments are underway to confirm the involvement of the other transcription factors predicted to regulate Ard1b transcription. Next we will examine coordination of DNA methylation and the binding of specific transcription factors in promotion of Ard1b transcription. On the other hand, the availability of Ard1b gene knockout mice enables us to study the role of Ard1b in testicular development as well as the biological significance of protein N-alpha acetylation. With a human cell culture model, we are investigating the difference in biological function between ARD1 and ARD1B, as well as, the global effect of deficiency of protein N-alpha acetylation on cellular function. Transcriptional regulation of Lin28 Investigators: Pang, Cho, Fang, Rennert;in collaboration with Chan Lin28 is a heterochronic gene involved in the temporal control of cell fate determination in C. elegans. It was shown to exert an enhancing effect on the reprogramming of somatic cells to embryonic stem (ES) cell-like state (i.e. iPS cells). In mammals, Lin28 is detected mostly in cells that possess proliferative/renewal capacity (e.g. embryonic stem cells, embryonal carcinoma cells, and mouse type A spermatogonia) and is absent in differentiated cells. The tissue-restricted and developmentally-regulated expression pattern of Lin28 suggests that its expression is subject to temporal and spatial regulation. Our preliminary data showed that Lin28 transcription is not regulated by DNA methylation;we hypothesized Lin28 transcription is primarily activated by the action of specific transcription factors that bind to its promoter. Reporter assays in mouse embryonal carcinoma cells P19 (Lin28-expressing) and mouse embryonic fibroblast cells NIH/3T3 (Lin28-non-expressing), led to localization of the core promoter of Lin28 gene - a region about 400 base-pairs upstream from its start codon. Specific modules of transcription factor binding sites have been predicted within this region, and by gene over-expression studies we have shown that at least Sp1 is able to activate Lin28 transcription in P19 cells. The involvement of other transcription factors in stimulating Lin28 expression, and the mechanism of suppression of Lin28 expression in non-expressing cells, are now under investigation. Effect of vitamin A deficiency on epigenomics of male germ cells: Investigators: Boucheron, Narani, Rennert;in collaboration with Chan This project studies the molecular consequences of vitamin A deficiency (VAD), in mice, on spermatogenesis. It utilizes expression profiling to study the genetic mechanism of vitamin A deficiency-induced arrest of spermatogonial stem cell differentiation, as well as the potential regeneration of spermatogenesis following restoration of vitamin A in the diet. The results will expand our knowledge of the molecular mechanisms of germ cell development. During the initial phase of the study we characterized the VAD experimental model by induction of the VAD diet from 7 weeks up to 28 weeks. At each time point measurement of vitamin A status included serum RBP (Retinol Binding Protein), and mRNA expression levels of retinoic acid (active metabolite of vitamin A) receptors in liver, brain and testis. In parallel we used histological techniques to characterize morphological changes in the testis. We compared the gene expression profile of control and VAD animals (18 weeks of deficiency) in an enriched population of spermatogonia. Our data confirmed the down-regulation of the retinoid pathways, and highlighted the importance in two other pathways, NF-B and -catenin. Future studies, will examine the effect of VAD on the epigenome of germ cells, and identify a set of genes responsible for VAD-induced male sterility. Effect of vitamin A deficiency on Sertoli cells: Sertoli cells, located in the seminiferous tubules, are essential to provide an adequate and protected environment for germ cell development. Sertoli cells from VAD animals lose the ability to adhere to the culture dishes, suggesting disruption of extracellular matrix and/or cell junctions. By real-time PCR, we documented VAD-induced decreased expression of GAP-43, a protein involved in the formation of the GAP junctions. Moreover PCR array analysis suggested deregulation of numerous genes of the extracellular matrix and/or cell junction. Literature reports identify a relationship between GAP-43, thyroid hormones and Sertoli cells differentiation. Hypothyroidism, associated with a decrease of GAP-43 expression, is associated with increased Sertoli cell proliferation and in the number of undifferentiated cells. We compared the number of Sertoli cells in the VAD animals, compared to controls with a cytoplasmic marker, vimentin, and by IHC observed a VAD-related increase of the number of Vimentin-positive cells. We probed the proliferation / differentiation status, using real-time PCR of these cells with Cyclin D2, as a marker of proliferation;AMH, as a marker of undifferentiation;and keratin 18, as a marker of differentiation. These studies utilize pure populations of Sertoli cells from Control and treated animals. We will also compare by the expression profile of the Sertoli cells from VAD and Control animals.
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